Cloning And Sequence Analysis Of E2 Gene Of HCV And Establishment Of Best Immuno-program Against HC

HC (hog cholera), the virulent infectious disease caused by HCV (hog cholera virus), had been controlled effectively by HCLV strain vaccine in the country. But atypical HC and inapparent HC began to appear after 1980s, which had resulted in great loss. The study is mainly about finding the reason that caused atypical HC through analysis and homology comparison on E2 gene sequences and amino acid sequences of E2 glycoprotein among strains HCLV, FC, HCV-JN, HCV-YC, SHIMEN et.al. At last, the best program against HC was established in order to control and erase HC.A pair of primers were chemically synthesized based on the cDNA sequence of hog cholera virus strain Brescia and used to amplify the cDNA fragments of envelope glycoprotein E2 gene of HCLV which coding the major protective antigen by reverse transcription-polymerase chain reaction (RT-PCR) from total intact RNA extracted from infected calf testicle cell culture by HCLV.Two cDNA fragments of the major antigenic protein E2 (gp55) gene of HCV-JN and HCV-YC strain were amplify by RT-PCR from total intact RNA extracted from the sixth passage infected pK-15 cell culture. The amplified E2 fragments of two HCV strains were all 1280bp in length by 2% agrose gel electrophoresis. Expected size of 1280bp of 2 fragments and their specificity were confirmed by restriction endonuclease digestion and then they were cloned respectively into pMD-18T vector. 1280bp cDNA sequence of two HCV E2 sequences sequenced and 426 residues amino acid sequences including E2 glycoprotein were deduced. Then the E2 gene nucleotide sequence and amino acid residues sequence were compared respectively with HCLV, strain FC, strain SHIMEN, strain Alfort of France, strain Brescia of Italy and so on. The result shows that the high degree of homology of E2 sequences and 426 residues amino acid sequencesexisted in the strain HCLV, HCV-JN, HCV-YC, FC and the epitopes between the four strains are not variable obviously by analysising the variation of some main amino acid residues substitutions of E2 major antigenic domains, confirming initially immunization with HCV vaccine strain can resist challenging of field strains.The sows, which are not pregnant, are vaccinated by HCV vaccine of different dose and then detected antibody against HCV with PPA-ELISA at different time. The results show that the high HCV antibody titer of sows last whole year and the program that the sows are vaccinated two times a year and four doses one time is the best program of sows. The mother-antibody levels of healthy unvaccinated piglets were tested with PPA-ELISA in different periods. The best time of first vaccination, which was established on basis of the result, was o day or 25th day of piglet. The immuno-antibody against HCV of healthy piglets, which had been vaccinated with HCLV strain vaccine in different period and dose, were detected by PPA-ELISA. The results showed that the immuno-protection were acquired by the immunized piglets which had been vaccinated 2 doses at o day or 4 doses at 25* day firstly and 4 doses at 60* day secondly. The 100% immuno-protection against HCV strain F114, V-JN and V-YC was observed in the experimental piglets that had been vaccinated with HCLV strain vaccine by the program above.